Tuberculosis (TB) is a major AIDS-related infection. The lengthy and cumbersome therapy currently available to treat TB has contributed to medical nonadherence and the emerging problems of multi-drug resistant (MDR)- and extensive-drug resistant (XDR)-TB. This prolonged therapy reflects the ability of Mycobacterium tuberculosis (Mtb) to persist in the infected host in a nonreplicating state characterized by antibiotic tolerance. The molecular mechanisms underlying Mtb growth restriction are unknown. We and others have shown that the alarmone hyperphosphorylated guanosine ((p)ppGpp) and the regulatory molecule inorganic polyphosphate (poly P) play a role in Mtb survival under growth-limiting conditions. However, the regulatory relationship between (p)ppGpp and poly P and the precise role of this network on Mtb growth restriction and antibiotic tolerance have not been elucidated. In this proposal, we plan to use Mtb recombinant strains conditionally overexpressing RelMtb, the stringent response enzyme responsible for (p)ppGpp synthesis, in order to test the hypothesis that (p)ppGpp is a molecular brake responsible for Mtb growth restriction and antibiotic tolerance. Next, using both poly P-deficient and poly P-accumulating Mtb recombinant strains, we will test the hypothesis that poly P regulates Mtb growth restriction and antibiotic tolerance. Finally, we will test the hypothesis that (p)ppGpp and poly P constitute a complex, feedback regulatory loop involving poly P- dependent expression of relMtb and (p)ppGpp-mediated inhibition of poly P hydrolysis. Although poly P is present in all cells, the highly-conserved bacterial enzyme responsible for poly P synthesis in Mtb has not been identified in mammalian cells, thus making it a potentially attractive target for drug development. A small molecule inhibitor of RelMtb would be predicted to lead to reduced Mtb synthesis of (p)ppGpp. Inhibition of this regulatory network may lead to reduced survival of persistent bacilli, with the potential to shorten the duration of TB chemotherapy. In addition to improving medical adherence and reducing the potential for the development of drug resistance, an abbreviated drug regimen to treat active TB could be especially useful in HIV co-infected patients, since drug-drug interactions and immune reconstitution may complicate the concurrent management of both infections.